Coronaviruses Lacking Exoribonuclease Activity Are Susceptible to Lethal Mutagenesis: Evidence for Proofreading and Potential Therapeutics

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Abstract
No therapeutics or vaccines currently exist for human coronaviruses (HCoVs). The Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) epidemic in 2002–2003, and the recent emergence of Middle East Respiratory Syndrome coronavirus (MERS-CoV) in April 2012, emphasize the high probability of future zoonotic HCoV emergence causing severe and lethal human disease. Additionally, the resistance of SARS-CoV to ribavirin (RBV) demonstrates the need to define new targets for inhibition of CoV replication. CoVs express a 3′-to-5′ exoribonuclease in nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication and is conserved across the CoV family. All genetic and biochemical data support the hypothesis that nsp14-ExoN has an RNA proofreading function. Thus, we hypothesized that ExoN is responsible for CoV resistance to RNA mutagens. We demonstrate that while wild-type (ExoN+) CoVs were resistant to RBV and 5-fluorouracil (5-FU), CoVs lacking ExoN activity (ExoN−) were up to 300-fold more sensitive. While the primary antiviral activity of RBV against CoVs was not mutagenesis, ExoN− CoVs treated with 5-FU demonstrated both enhanced sensitivity during multi-cycle replication, as well as decreased specific infectivity, consistent with 5-FU functioning as a mutagen. Comparison of full-genome next-generation sequencing of 5-FU treated SARS-CoV populations revealed a 16-fold increase in the number of mutations within the ExoN− population as compared to ExoN+. Ninety percent of these mutations represented A:G and U:C transitions, consistent with 5-FU incorporation during RNA synthesis. Together our results constitute direct evidence that CoV ExoN activity provides a critical proofreading function during virus replication. Furthermore, these studies identify ExoN as the first viral protein distinct from the RdRp that determines the sensitivity of RNA viruses to mutagens. Finally, our results show the importance of ExoN as a target for inhibition, and suggest that small-molecule inhibitors of ExoN activity could be potential pan-CoV therapeutics in combination with RBV or RNA mutagens. RNA viruses have high mutation rates (10−3 to 10−5 mutations/nucleotide/round of replication), allowing for rapid viral adaptation in response to selective pressure. While RNA viruses have long been considered unable to correct mistakes during replication, CoVs such as SARS-CoV and the recently emerged MERS-CoV are important exceptions to this paradigm. All CoVs encode an exoribonuclease activity in nonstructural protein 14 (nsp14-ExoN) that is proposed to prevent and/or remove misincorporated nucleotides. Because of the demonstrated resistance of SARS-CoV to the antiviral drug ribavirin (RBV), we hypothesized that ExoN is responsible for CoV resistance to RNA mutagens. Using RBV and the RNA mutagen 5-fluorouracil (5-FU), we show that CoVs lacking ExoN activity (ExoN−) are highly susceptible to RBV and 5-FU, in contrast to wild-type (ExoN+) CoVs. The inhibitory activity of 5-FU against ExoN− viruses resulted specifically from 5-FU incorporation during viral RNA synthesis that lead to extensive mutagenesis within the viral population, and was associated with a profound decrease in virus specific infectivity. These results demonstrate the proofreading activity of ExoN during virus replication and suggest that inhibitors of ExoN activity could be broadly useful inhibitors of CoV replication in combination with RBV or RNA mutagens.